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Nucleic Acids Research, 1985, Vol. 13, No. 12 4343-4363
© 1985

Articles

Transition of a cloned d(AT)n-d(AT)n tract to a cruciform in vivo

David B. Haniford and David E. Pulleyblank

Department of Biochemistry, University of Toronto Toronto, Ontario M5S-1A8, Canada

Received March 14, 1985. Revised May 29, 1985. Accepted May 29, 1985.

A 34 base pair tract of the simple repeating dinucleotide d(AT)n-d(AT)n cloned into a 2.4 kb polylinker plasmid vector undergoes a structural transition in response to negative superhelical coiling. The transition has been characterized by 2 dimensional gel electrophoresis, mapping of Sl, Pl and T7 endonuclease 1 sensitive sites, and mapping of sites that are sensitive to modification by bromoacetaldehyde. After S1 nuclease treatment it is possible to trap supercoiled species that are nicked on one or both strands near the center of the palindrome. These data show that the alternate state adopted by the d(AT)n-dAT)n insert is a cruciform rather than a Z conformation. Unlike other B-cruciform transitions the transition in d(AT)n-d(AT)n has a low activation energy and the transition is facilitated by the presence of magnesium ions. Evidence from in-vivo topoisomer distributions is presented which shows that under conditions of blocked protein synthesis the d(AT)n-d(AT)n insert will spontaneously adopt the cruciform state in-vivo in E. coli


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